The invention relates to fixed arrays of nucleic acid-protein fusions and, in particular, RNA-protein fusions, on solid supports.
Certain macromolecules, such as proteins, are known to interact specifically with other molecules based on the three-dimensional shapes and electronic distributions of those molecules. For example, proteins interact selectively with other proteins, with nucleic acids, and with small-molecules. Modern pharmaceutical research relies on the study of these interactions; the development of new drugs depends on the discovery of compounds that bind specifically to biologically important molecules.
The discovery of a single drug candidate can require the screening of thousands of compounds. It is therefore important to be able to screen large numbers of compounds rapidly and efficiently. One method for screening a large number of compounds is to fix possible binding partners, such as proteins, to a solid support.
It is difficult to prepare arrays of isolated proteins on solid supports, however, for a variety of reasons. First of all, proteins cannot always be easily attached to the planar surfaces traditionally used to make other fixed arrays, such as nucleic acid microchips. More importantly, because proteins can interact with the functional groups on the surfaces of these supports, the proximity of the protein to the surface can lead to disruption of the protein structure.
In general, the invention features a solid support including an array of immobilized capture probes; each of the capture probes includes a non-nucleosidic spacer group and an oligonucleotide sequence to which a nucleic acid-protein fusion is bound (for example, hybridized or covalently bound). In preferred embodiments, the nucleic acid-protein fusion is an RNA-protein fusion, and the protein component is encoded by the nucleic acid (for example, the RNA). The spacer group can include a polyalkylene oxide, for example, polyethylene oxide. A preferred spacer group includes hexaethylene oxide. The capture probe may also include a photocleavable linker.
The oligonucleotide sequence can include a modified base, such as 5-propyne pyrimidine. It can also include an internucleotide analog (such as 3xe2x80x2-phosphoramidate) or a carbohydrate modification (such as a 2xe2x80x2-O-methyl group). The nucleic acid-protein fusion can include a hybridization tag sequence. The hybridization tag sequence can also include a modified base, an internucleotide analog, or a carbohydrate modification.
In a preferred embodiment, the capture probe further includes a reactive moiety (for example, a nucleophilic group), such as a primary amino group. In another preferred embodiment, the nucleic acid-protein fusion is covalently linked to the capture probe (for example, by photo-crosslinking); in one preferred approach, this is accomplished by including one or more psoralen moieties in the capture probe or in the capture probe-fusion hybridization reaction mixture. A preferred solid support is a glass or silica-based chip.
In a related aspect, the invention features a solid support including an array of immobilized capture probes; each of the capture probes is attached to the surface of the solid support through a non-nucleosidic spacer group, and each of the capture probes includes an oligonucleotide sequence to which a nucleic acid-protein fusion (for example, an RNA-protein fusion) is bound (for example, hybridized or covalently bound).
In another related aspect, the invention features a solid support including an array of immobilized capture probes; each of the capture probes includes a non-nucleosidic spacer group and an oligonucleotide sequence to which a ribosome display particle is bound (for example, hybridized or covalently bound).
In yet another related aspect, the invention features a method for preparing a solid support. The method includes the steps of: (a) preparing a capture probe by linking a spacer group to an oligonucleotide sequence; (b) attaching the capture probe to the solid support; and (c) binding (for example, hybridizing or covalently binding) a nucleic acid-protein fusion (for example, an RNA-protein fusion) to the capture probe.
The invention also features a second general method for preparing a solid support. This method includes the steps of: (a) attaching a spacer group to a surface of the solid support; (b) attaching a bifunctional linker to the spacer group; (c) attaching a capture probe to the bifunctional linker; and (d) binding (for example, hybridizing or covalently binding) a nucleic acid-protein fusion (for example, an RNA-protein fusion) to the capture probe.
In a second aspect, the invention features a method for detecting an interaction between a protein and a compound. The method includes the steps of: (a) providing a solid support including an array of immobilized capture probes, where each of the capture probes includes a non-nucleosidic spacer group and an oligonucleotide sequence to which a nucleic acid-protein fusion is bound (for example, hybridized or covalently bound); (b) contacting the solid support with a candidate compound under conditions which allow an interaction between the protein portion of the nucleic acid-protein fusion and the compound; and (c) analyzing the solid support for the presence of the compound as an indication of an interaction between the protein and the compound.
Alternatively, the invention features another method for detecting an interaction between a protein and a compound; this method involves the steps of: (a) providing a population of nucleic acid-protein fusions; (b) contacting the population of nucleic acid-protein fusions with a candidate compound under conditions which allow an interaction between the protein portion of the nucleic acid-protein fusion and the compound; (c) contacting the product of step (b) with a solid support that includes an array of immobilized capture probes, each of the capture probes including a non-nucleosidic spacer group and an oligonucleotide sequence to which a nucleic acid-protein fusion binds (for example, hybridizes or covalently binds); and (d) analyzing the solid support for the presence of the compound as an indication of an interaction between the protein and the compound.
In a preferred embodiment of each of the above methods, the nucleic acid-protein fusion is an RNA-protein fusion. In another preferred embodiment, the compound is labeled. Compounds that can be screened using these methods include, without limitation, proteins, drugs, therapeutics, enzymes, and nucleic acids.
In a third aspect, the invention features an array (for example, an addressable array) of nucleic acid-protein fusions including at least 102 different fusions/cm2. Preferably, the nucleic acid-protein fusions are RNA-protein fusions, and the array includes at least 104 different fusions/cm2.
In a related aspect, the invention features a method for generating an addressable array of molecules. The method involves: (a) providing a solid support on which an array of nucleic acid molecules is immobilized; (b) contacting the solid support with a population of addressable molecules; and (c) allowing the addressable molecules to orient themselves on the solid support by sequence-dependent recognition and binding of the immobilized nucleic acid molecules.
In preferred embodiments of this method, the addressable array of molecules is an array of nucleic acid-protein fusions (for example, an array of RNA-protein fusions); the addressable molecules orient themselves on the solid support by base pairing (for example, hybridization) with the immobilized nucleic acid molecules; the solid support is a glass or silica-based chip; and the nucleic acid molecules immobilized on the solid support are capture probes, each including a non-nucleosidic spacer group and an oligonucleotide sequence to which the addressable molecule binds.
As used herein, by an xe2x80x9carrayxe2x80x9d is meant a fixed pattern of immobilized objects on a solid surface or membrane. Typically, the array is made up of nucleic acid-protein fusion molecules (for example, RNA-protein fusion molecules) bound to capture nucleic acid sequences which themselves are immobilized on the solid surface or membrane. The array preferably includes at least 102, more preferably at least 103, and most preferably at least 104 different fusions, and these fusions are preferably arrayed on a 125xc3x9780 mm, and more preferably on a 10xc3x9710 mm, surface. By an xe2x80x9caddressable arrayxe2x80x9d is meant that the locations, or addresses, on the solid support of the members of the array (for example, the nucleic acid-protein fusions) are known; the members of the array are referred to as xe2x80x9caddressable moleculesxe2x80x9d and are utilized in methods for screening for subsequent molecular interactions (for example, for screening for interactions between the addressable nucleic acid-protein fusions and candidate therapeutics).
By xe2x80x9cnucleic acid-protein fusionxe2x80x9d is meant a nucleic acid covalently bound to a protein. By xe2x80x9cnucleic acidxe2x80x9d is meant any two or more covalently bonded nucleotides or nucleotide analogs or derivatives. As used herein, this term includes, without limitation, DNA, RNA, and PNA. By xe2x80x9cproteinxe2x80x9d is meant any two or more amino acids, or amino acid analogs or derivatives, joined by peptide or peptoid bond(s), regardless of length or post-translational modification. As used herein, this term includes, without limitation, proteins, peptides, and polypeptides.
By xe2x80x9chybridization tagxe2x80x9d is meant a non-coding oligonucleotide sequence that differs sufficiently in sequence from other nucleic acid sequences in a given population or reaction mixture that significant cross-hybridization does not occur. When multiple hybridization tags are utilized in a single reaction mixture, these tags also preferably differ in sequence from one another such that each has a unique binding partner under the conditions employed.
By a xe2x80x9cpopulationxe2x80x9d is meant more than one molecule.
By a xe2x80x9csolid supportxe2x80x9d is meant any solid surface including, without limitation, any chip (for example, silica-based, glass, or gold chip), glass slide, membrane, bead, solid particle (for example, agarose, sepharose, or magnetic bead), column (or column material), test tube, or microtiter dish.